Recent studies in the murine system have shown that CD4+ T helper (Th) cells can often be subdivided based upon profiles of cytokines produced, and convincing evidence is now at hand for Th cell subsets in humans with autoimmune, allergic and infectious diseases. Two subsets, Th1 and Th2 (type 1 and type 2) are best characterized and type 1 Th cells produce interleukin-2 (IL-2), interferon gamma (IFN-gamma) and TNF-beta for cell- mediated immunity (CMI) in order to protect against intracellular parasites. Type 2 Th cells produce IL-4, IL-5, IL-6 and IL-10 and upregulate IgG subclass, IgE and IgA antibody responses. Important recent studies have shown that after HIV infection, the progression to AIDS is characterized by loss of Th1 cells and with increases in Th2 cells which are associated with well documented increases in serum IgG and IgA levels. Our own studies have recently shown that oral immunization induced mainly Th2-type cells for regulation of mucosal secretory IgA (S-IgA) responses. Thus, we have postulated that mucosal vaccines should induce Th2 cells for optimal S-IgA responses and Th1-type cells for CMI and help for CD8+ cytotoxic T lymphocyte (CTL) responses. We have further hypothesized that SIV infection results in loss of TH1 cells in systemic tissues; however mucosal CD4+ Th2 cell responses may remain functional. Further, this hypothesis would suggest that SIV/HIV vaccines should optimize induction of Th2 cells for mucosal antibodies as well as type 1 cells for systemic CMI and CTL responses. To test this, our grant has been divided into two parts. In the first part, we will assess Th1 and Th2 cell imbalances in both systemic and mucosa-associated tissues of SIV infected monkeys. We will use single-cell assays, i.e., enzyme-linked immunospots (ELISPOTs) to assess Th1 (IFN-gamma and IL-2) and Th2(IL04 and IL-10)cytokines as well as RNA protection assay (RPA) and reverse transcriptase (RT)-PCR for analysis of cytokine mRNA. The first specific aim will focus on peripheral blood CD4+T cells for memory (recall) type 1 or type 2 responses to standard vaccines and to mitogen (PHA). The second specific aim will continue to assess Th1/Th2 imbalances in SIV infection, but will emphasize CD4+ Th cell responses to recall vaccine, to PHA and to SIV components with T lymphocytes isolated from both mucosa-associated and systemic lymphoid tissues. The overall emphasis in the second part of this grant will be to assess optimal Th1 and Th2 cell responses following deliberate mucosal immunization with whole SIV or its components (rgp130 and gp27). We plan state-of-the-art mucosal immunization strategies using cholera toxin (CT) and with SIV gp130 components conjugated to CT-B as adjuvant as well water- based microspheres and recombinant bacteria (rSalmonella tryphimurium and rVibrio cholerae) for mucosal delivery of SIV components. In specific aim 3, we will use CT as adjuvant to optimize mucosal immune responses including induction of type 1 and especially type 2 CD4+ responses. In the final aim, we plan to compare other delivery systems, i.e., water-based microspheres and recombinant bacteria for mucosal delivery. This effort is part of a Collaboration with four other groups with expertise in heterosexual (CRPRC) and homosexual (Guy's/CAMR) SIV infection and immunity, with experience in mucosal vector systems (VRI) and with unique models to study S-gA antibody function, in vitro (CWR). Together we can approach the goal of inducing effective mucosal and systemic immunity fy using appropriate cellular and molecular techniques developed for rhesus macaques.